Method and apparatus for spatial control of cellular growth

ABSTRACT

A three-dimensional cell growth containment article is described, which includes a molded body channelized by removal of sacrificial channelizing element(s) therefrom, so that the molded body contains one or more channel(s) therein, with a matrix material in at least one of such channel(s) that is supportive of three-dimensional cell growth in the matrix material. A method for making such articles is also described, in which a molded body is formed with one or more sacrificial channelizing element(s) therein, following which the sacrificial channelizing element(s) are removed. The three-dimensional cell growth containment articles of the present disclosure may be utilized in any applications in which there exists a need to reproducibly generate three-dimensional cellular structures, e.g., islet transplantation for diabetes treatment, transplantation of hormone secreting cells, cellular scaffolds for wound healing, and generation of tissue engineering structures to regain structural usefulness for orthopedic applications.

CROSS-REFERENCE TO RELATED APPLICATION

The benefit under 35 USC § 119 of U.S. Provisional Patent Application62/771,958 filed Nov. 27, 2018 in the names of Leah Marie Johnson,Ninell Pollas Mortensen, Ginger Denison Rothrock, and Nicolas D. Huffmanfor METHOD AND APPARATUS FOR SPATIAL CONTROL OF CELLULAR GROWTH ishereby claimed. The disclosure of U.S. Provisional Patent Application62/771,958 is hereby incorporated herein by reference, in its entirety,for all purposes.

FIELD

The present disclosure relates to three-dimensional cell growthcontainment articles useful in retentive deployment of cells, e.g., forculturing of cell populations to produce secreted therapeutic agents,and to methods of making and using such articles.

DESCRIPTION OF THE RELATED ART

In many biological fields of endeavor, there exists a need to provide acellular population whose growth is controllably managed and maintained.In specific, there is a need for temporally and spatially controllingcell growth in a wide variety of applications.

An ability to spatially and temporally guide cellular growth affordsvast possibilities in research as well as clinical applications, infields as diverse as tissue engineering, therapeutic treatment andprevention, biomechanics, cellular biology, and cell signaling. Cellularviability and proliferation depends on a multitude of conditions,including for example oxygenation, nutrient accessibility, cellularcues, and biocompatible environments.

Typically, three-dimensional growth of cellular cultures can be carriedout using a polymeric matrix, in which the cells are seated onto thematrix and provide cues to guide cellular proliferation. Cells can beseated directly onto pre-form polymers, or alternatively incorporatedinto a monomer mixture prior to polymerization using in-situ cellculture methods. Polymer matrices may for example be constituted byhydrogels that can be fabricated using light-initiated free radicalcross-linking, Michaels-type addition cross-linking, or redox-initiatedcross-linking techniques, or ionic cross-linking methods such as the useof alginate materials that cross-link utilizing divalent cations (e.g.,Ca⁺²).

Three-dimensional platforms for cells typically rely on inherentphysicochemical properties of the polymerization process to generatespecific geometries. For example, alginate may be added drop-wise to asolution of cross-linker material, resulting in spherical alginatematrices on which cells are seated. Other approaches for generatingthree-dimensional cellular cultures with a specific geometry utilizepolymerization techniques, such as guided light irradiation during photopolymerization to form specific supporting matrix architectures. Inthese efforts, polymer properties determine mechanical stability,biocompatibility, transport, and other characteristics that directlyaffect the ability to maintain cellular growth.

In consequence, the art continues to seek improvements in achievingcontrolled spatial arrangements of cells, to achieve desirablethree-dimensional formations of cellular populations that are createdand maintained for their desired purposes, such as investigativeefforts, therapeutic interventions, drug development, etc.

SUMMARY

The present disclosure relates to a three-dimensional cell growthcontainment articles and to methods of making and using such articles.

In one aspect, the disclosure relates to a method of making athree-dimensional cell growth containment article, such methodcomprising: providing a separable mold body defining a mold cavitytherewithin, the separable mold body comprising engageable mold bodyportions; engaging the engageable mold body portions with one another toconstitute the separable mold body, with at least one sacrificialchannelizing element compressively retained between the engaged moldbody portions so that the sacrificial channelizing element spans themold cavity; introducing a curable medium into the mold cavity so thatthe curable medium fills the mold cavity to a predetermined extent, andcontacts and circumscribes the sacrificial channelizing element(s) in abulk volume of the curable medium; at least partially curing the curablemedium to form an at least partially cured article; and removing thesacrificial channelizing element(s) from the at least partially curedarticle so that the at least partially cured article is channelized bythe removed sacrificial channelizing element(s), to yield thethree-dimensional cell growth containment article containing channel(s)for three-dimensional cell growth therein.

In another aspect, the disclosure relates to a three-dimensional cellgrowth containment article, comprising a molded body channelized byremoval of sacrificial channelizing element(s) therefrom, so that themolded body contains one or more channel(s) therein, with a matrixmaterial in at least one of said channel(s) that is supportive ofthree-dimensional cell growth in the matrix material.

Other aspects, features and embodiments of the disclosure will be morefully apparent from the ensuing description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic representation of a mold usefully employed forforming a three-dimensional cell growth containment article inaccordance with one embodiment of the present disclosure.

FIG. 2 schematically illustrates the removal of the sacrificial coreelement from a molded body (polymer structure) to form a through-boreopening as a hollow core passage in the polymer structure of the moldedbody in the formation of a three-dimensional cell growth containmentarticle according to one embodiment of the present disclosure.

FIG. 3 depicts cross sections of various illustrative polymer structuresthat may be fabricated with correspondingly configured molds for makingthree-dimensional cell growth containment articles of the presentdisclosure.

FIG. 4 is a digital camera image of an aluminum mold that was used toprepare a cylindrical cell growth containment article with a hollowcore.

FIG. 5 shows CAD drawings of the mold design of the mold of FIG. 4 .

FIG. 6 shows digital camera images of the mold of FIGS. 4 and 5 .

FIG. 7 is a microscopic image of a hollow polymeric cylinder comprisingpolyacrylamide and non-cross-linked alginate with a hollow core.

FIG. 8 is a microscopic image of a cross-section of the cylindricalpolymer structure comprising polyacrylamide and alginate, and having ahollow core passage with a diameter of 1 mm.

FIG. 9 is a digital camera image of a cylindrical polymer structure of apolyacrylamide/alginate polymer containing a hollow core passage, inwhich the structure was 12 mm in length with an outer diameter of 2 mm.

FIG. 10 summarizes the steps involved in forming a hollow corecylindrical polymer structure of a three-dimensional cell growthcontainment article, according to one embodiment of the disclosure.

FIG. 11 shows a sequence of 3 steps for producing a three-dimensionalcell growth containment article including a core of cells in athree-dimensional scaffold.

FIG. 12 shows the results after culturing Raji cells in athree-dimensional cell growth containment article for 3 days, influorescence images of Raji cells in the interior of the article, asstained with a Live-Dead staining kit.

FIG. 13 shows RIN-5F cells alive and proliferating in athree-dimensional cell growth containment article after 25 days.

FIG. 14 shows steps for loading a cell mixture into a three-dimensionalcell growth containment article, using a solution representing asurrogate for cells for purposes of illustration.

DETAILED DESCRIPTION

The present disclosure relates to three-dimensional cell growthcontainment articles useful in retentive deployment of cells, e.g., forculturing of cell populations to produce secreted therapeutic agents,and to methods of making and using such articles.

The present inventors have devised a highly efficient manner ofprecisely depositing cells in a controlled spatial arrangement, andeffecting three-dimensional cellular growth, by the provision ofretentive structural articles accommodating such deposition and growth,as hereinafter more fully described with respect to such articles andtheir methods of manufacture and use.

Three-dimensional cell growth containment articles of the presentinvention enable spatial arrangements of multiple cell types withvarious polymeric matrices. Such three-dimensional cell growthcontainment articles are amenable to ready fabrication by use of moldingtechniques as hereinafter more specifically described, in which moldscan be customized with different geometries, injection ports, polymericmaterials of construction, and sizes and dimensional characteristics, tosupport three-dimensional cellular growth in an efficient and effectivemanner.

Molds utilized for such purpose in accordance with the presentdisclosure may have multi-compartmental sections to enable injection ofdifferent solutions containing different cell types, different monomers,and different pre-polymers. Such molds may be formed with troughs toaccommodate and hold injected solutions, with the troughs being ofvarious shapes and/or orientations as desired, e.g., cylindricaltroughs. The molds can accommodate sacrificial materials to serve astemplates for generating polymeric structures with voids. Alternatively,the mold itself can exist as a sacrificial template, wherein the molddegrades after polymerization of the injected solutions. The mold canaccommodate a variety of types of monomers and pre-polymers andmechanisms for polymerization, including ionic cross-linking andcovalent cross-linking, such as chain polymerization and steppolymerization mechanisms.

In the ensuing description of the three-dimensional cell growthcontainment article of the present disclosure and the methods of makingand using same, it will be recognized that the article may be fabricatedand the methods may be performed utilizing any of a variety of features,elements, techniques, and approaches, as described hereafter, and thatthese various features, elements, techniques, and approaches may beselectively implemented in any suitable implementations and combinationsthereof as may be appropriate in a given application of the articles andmethods of the present disclosure.

The three-dimensional cell growth containment articles of the presentdisclosure may be utilized in any applications in which there exists aneed to reproducibly generate three-dimensional cellular structures,e.g., islet transplantation for diabetes treatment, transplantation ofhormone secreting cells, cellular scaffolds for wound healing,generation of tissue engineering structures to regain structuralusefulness for orthopedic applications, etc. The three-dimensional cellgrowth containment articles of the present disclosure may be constructedof a wide variety of materials in a wide variety of conformations,appropriate to the specific cellular material present in such articles.For example, the three-dimensional cell growth containment article maybe provided of a permeable material enabling oxygen and nutrientdiffusion into the passage or compartment containing the cells, and thecomposition and conformation of the article may otherwise be designedfor maintaining viability and growth of the cells contained in thearticle.

The disclosure in one aspect relates to a method of making athree-dimensional cell growth containment article, such methodcomprising: providing a separable mold body defining a mold cavitytherewithin, the separable mold body comprising engageable mold bodyportions that may be mated with one another to cooperatively form themold body; engaging the engageable mold body portions with one anotherto constitute the separable mold body, with at least one sacrificialchannelizing element compressively retained between the engaged moldbody portions so that the sacrificial channelizing element spans themold cavity; introducing a curable medium into the mold cavity so thatthe curable medium fills the mold cavity to a predetermined extent, andcontacts and circumscribes the sacrificial channelizing element(s) in abulk volume of the curable medium; at least partially curing the curablemedium to form an at least partially cured article; and removing thesacrificial channelizing element(s) from the at least partially curedarticle so that the at least partially cured article is channelized bythe removed sacrificial channelizing element(s), to yield thethree-dimensional cell growth containment article containing channel(s)for three-dimensional cell growth therein. The method described in thisparagraph is hereafter referred to as the “broadly described method ofthe disclosure” and any of the features and aspects hereinafterdescribed for such broadly described method of the disclosure may beselectively aggregated with any other features and aspects described forsuch broadly described method of the disclosure.

In the broadly described method of the disclosure, the curable mediummay be of any suitable type, and may for example comprise an alginate,cellulose, chitosan, collagen, fibrin, glycosaminoglycan,carboxymethylcellulose, a monomer or pre-polymer containing a cellattachment signal agent, acrylate, methacrylate, acrylamide, materialthat is permeable to cellular nutrients when cells are disposed in thechannel(s) of the three-dimensional cell growth containment article,growth factor, hydrogel material, or a combination of two or more of theforegoing. In specific embodiments, the curable medium may comprisepolyethylene glycol diacrylate or polyethylene glycol dimethacrylate,e.g., wherein the polyethylene glycol chain has a molecular weight(number average molecular weight) in a range of from 1,000 to 5,000.

A curable medium may be utilized in the broadly described method of thedisclosure, wherein the curable medium comprises an alginate having aG:M ratio of guluronic acid (G):mannuronic acid (M) that is greater than1.5. The at least partially cured curable medium in the broadlydescribed method of the disclosure may for example comprisepolylactic-co-glycolic acid (PLGA), polycaprolactone, polyethyleneglycol, polylactide, polyglycolide, ethylene-vinyl acetate copolymer,polyvinylalcohol (PVA), or a combination of two or more of theforegoing. In various other embodiments, the at least partially curedcurable medium may comprise a natural polymer, such as collagen, fibrin,chitosan, glycosaminoglycan, or combinations of two or more of theforegoing. As another variant of the broadly described method of thedisclosure, the at least partially cured curable medium may comprise animmunoprotective polymer.

The curable medium in the broadly described method of the disclosure maycomprise a monomer or pre-polymer containing a cell attachment signalagent, e.g., a cell attachment signal agent comprisingarginylglycylaspartic acid, or such agent may be added as a part ofsubsequently introduced fill material to the channel(s) of the article.In other embodiments, the curable medium may comprise a growth factor,such as for example a vascular endothelial growth factor (VEGF) or afibroblastic growth factor, or such factor or factors may be added as apart of subsequently introduced fill material to the channel(s) of thearticle. It will be recognized that in the three-dimensional cell growthcontainment article of the present disclosure and its associated methodsof manufacture and use, the curable medium, corresponding curedmaterial, and fill materials may generally comprise any of the specificingredients variously described herein in connection with specificembodiments of the curable medium, corresponding cured material, or fillmaterial, which are compatible with the article and its methods ofmanufacture and use. Such ingredients may for example includecell-signaling agents, growth factors, siRNAs, and any other suitableingredients that control, sustain, mediate, or otherwise affect cells inthe three-dimensional cell growth containment article. In a specificimplementation, the method of the disclosure may comprise introducing afill material to the channel(s) of the three-dimensional cell growthcontainment article, wherein the fill material comprises adherent cellsin combination with monomers or pre-polymers that contain a cellattachment signal agent, polymerizing the monomers or pre-polymers toform a cell support matrix whereon the adherent cells attach by actionof the cell attachment signal agent, and contacting the cells supportedin the cell support matrix with a cell culture medium to producethree-dimensional growth of the cells in the cell support matrix.

In the broadly described method of the disclosure, the three-dimensionalcell growth containment article may contain multiple channels. At leastsome of such multiple channels may be in cell-signaling proximity to oneanother. The at least partially cured curable medium in the broadlydescribed method of the disclosure may comprise material that ispermeable to cellular nutrients when cells are disposed in thechannel(s) of the three-dimensional cell growth containment article.

In one embodiment of the broadly described method of the disclosure, thecurable medium comprises a hydrogel material. The hydrogel material maybe ionically cross-linkable, and may for example comprise an alginate.The alginate may be cross-linkable with divalent cations, such ascalcium (Ca⁺²) cations. Accordingly, the broadly described method of thedisclosure may be carried out, as comprising introducing into one ormore of the channel(s) a mixture of cells and alginate, followed bycontacting the three-dimensional cell growth containment article with across-linking solution of calcium dichloride or aluminum trichloride tocross-link the alginate in the mixture of cells and alginate, so thatthe cells are supported in a matrix of cross-linked alginate, andcontacting the cells supported in the matrix of cross-linked alginate inthe three-dimensional cell growth containment article, with a cellculture medium to produce three-dimensional growth of the cells in thematrix.

The broadly described method of the disclosure may be carried out,wherein the engageable mold body portions are formed of degradablesacrificial material, and the method further comprises degrading thedegradable sacrificial material after at least partially curing thecurable medium to form the at least partially cured article. In suchmethod, the degrading may comprise at least one of chemicallydecomposing the degradable sacrificial material, volatilizing thedegradable sacrificial material, dissolving the degradable sacrificialmaterial, irradiating the degradable sacrificial material withdegradative radiation, and thermally decomposing the degradablesacrificial material. In a specific embodiment, the degradablesacrificial material may be chemically decomposed by hydrolysis thereof.

In various embodiments of the broadly described method of thedisclosure, the separable mold body may comprise a port for introducingthe curable medium into the mold cavity.

The curable medium and in the broadly described method of the disclosuremay be at least partially cured by a curing modality including at leastone of ionic cross-linking, covalent cross-linking, hydrolysis, andpolymerization.

The sacrificial channelizing element in the broadly described method ofthe disclosure may comprise a fiber, filament, or cord that ismaintained in tension across its spanning extent. The mold cavity may becylindrical, with the sacrificial channelizing element extending along acenterline of the cylindrical mold cavity.

The broadly described method of the disclosure may be carried out withthe additional step of introducing a fill material to the channel(s) ofthe three-dimensional cell growth containment article. The fill materialmay comprise at least one of monomer, prepolymer, cells, growth factors,nano materials, small molecule drugs, antibodies, proteins, nucleicacids, nutrients, tracers, bacteria, viruses, and gas-producingsubstances. In various embodiments, the fill material may comprise abiological material, which may comprise naturally occurring biologicalmaterial and/or synthetic biological material. The biological materialmay for example comprise at least one of cells, antibodies, proteins,nucleic acids, bacteria, and viruses. In various embodiments, the fillmaterial comprises cells, e.g., suspension cells, or adherent cellsaffixed to interior surface of the channel(s) or affixed to materialscomprising or comprised in the at least partially cured medium of thethree-dimensional cell growth containment article, or affixed to hormonesecreting cells. The cells may be of one or more cell types ofcorticotropes, gonadotropes, thyrotropes, lactotropes, somatotropes,magnocellular neurosecretory, thyroid epithelial, parafollicular,parathyroid, adrenal gland, macula densa kidney, Leydig, granulosalutein, theca lutein, peripolar kidney, and mesangial kidney cells. Thecells in other embodiments may comprise islets of Langerhans, e.g., oneor more cell types of alpha cells, beta cells, delta cells, PP cells,and Epsilon cells. In still other embodiments, the cells may compriseliver cells, e.g., of one or more cell types of hepatocytes, stellatecells, Kupfer cells, and liver endothelial cells. In still otherembodiments, the cells may comprise peptide hormone secreting cells,e.g., wherein the peptide hormone secreting cells are one or more celltypes of alpha cells, beta cells, corticotropic cells, delta cells,gonadotropin cells, gastric chief cells, lactotropic cells,parafollicular cells, parathyroid cells, somatotropic cells, andthyrotropic cells.

In various embodiments of the broadly described method of thedisclosure, the fill material comprises adherent cells in combinationwith monomers or pre-polymers that contain a cell attachment signalagent, e.g., a cell attachment signal agent comprisingarginylglycylaspartic acid. In other embodiments, the curable medium maycomprise a growth factor, such as for example a vascular endothelialgrowth factor (VEGF) or a fibroblastic growth factor.

In various embodiments of the broadly described method of thedisclosure, the curable material comprises acrylamide, water, alginate,ammonium persulfate, and tetramethylenediamine (TEMED). Such curablematerial may be cured at room temperature to polymerize the acrylamideto polyacrylamide to form the at least partially cured articlecomprising polyacrylamide and non-cross-linked alginate. The method maycomprise contacting the at least partially cured article comprisingpolyacrylamide and non-cross-linked alginate with a curing solution ofcalcium dichloride or aluminum trichloride to cross-link the alginate,to yield the three-dimensional cell growth containment article as apolymeric structure comprising polyacrylamide and alginate polymer. Themethod may further comprise sterilizing the three-dimensional cellgrowth containment article, e.g., by contacting the three-dimensionalcell growth containment article with a sterilizing medium, such asalcohol. The method may further comprise conditioning thethree-dimensional cell growth containment article in cell media.

The broadly described method of the disclosure may further compriseintroducing a fill material to the channel(s) of the three-dimensionalcell growth containment article, wherein the fill material comprisesadherent cells in combination with monomers or pre-polymers that containa cell attachment signal agent, polymerizing the monomers orpre-polymers to form a cell support matrix whereon the adherent cellsattach by action of the cell attachment signal agent, and contacting thecells supported in the cell support matrix with a cell culture medium toproduce three-dimensional growth of the cells in the cell supportmatrix. In another implementation, the broadly described method of thedisclosure may further comprise introducing into one or more of thechannel(s) a mixture of cells and alginate, followed by contacting thethree-dimensional cell growth containment article with a cross-linkingsolution of calcium dichloride or aluminum trichloride to cross-link thealginate in the mixture of cells and alginate, so that the cells aresupported in a matrix of cross-linked alginate. The method may furthercomprise contacting the cells supported in the matrix of cross-linkedalginate in the three-dimensional cell growth containment article, witha cell culture medium to produce three-dimensional growth of the cellsin the matrix.

The present disclosure in another aspect relates to a three-dimensionalcell growth containment article, comprising a molded body channelized byremoval of sacrificial channelizing element(s) therefrom, so that themolded body contains one or more channel(s) therein, with a matrixmaterial in at least one of said channel(s) that is supportive ofthree-dimensional cell growth in the matrix material. The articledescribed in this paragraph is hereafter referred to as the “broadlydescribed article of the disclosure” and any of the features and aspectshereinafter described for such broadly described article of thedisclosure may be selectively aggregated with any other features andaspects described for such broadly described article of the disclosure.

In the broadly described article of the disclosure, the molded body maycomprise a cured material derived from alginate, cellulose, chitosan,carboxymethylcellulose, acrylate, methacrylate, acrylamide, or acombination of two or more of the foregoing. The molded body may forexample comprise a cured material derived from polyethylene glycoldiacrylate or polyethylene glycol dimethacrylate, e.g., wherein thepolyethylene glycol chain has a molecular weight (number averagemolecular weight) in a range of from 1,000 to 5,000.

In various embodiments of the broadly described article of thedisclosure, the molded body may comprise a cured material derived froman alginate having a G:M ratio of guluronic acid (G):mannuronic acid (M)that is greater than 1.5.

In various embodiments of the broadly described article of thedisclosure, the molded body may comprise polylactic-co-glycolic acid(PLGA), polycaprolactone, polyethylene glycol, polylactide,polyglycolide, ethylene-vinyl acetate copolymer, or a combination of twoor more of the foregoing.

In various embodiments of the broadly described article of thedisclosure, the molded body may comprise an immunoprotective polymer.

The broadly described article of the disclosure in various embodimentsmay have the molded body comprising a cured material derived from amonomer or pre-polymer containing a cell attachment signal agent, e.g.,arginylglycylaspartic acid. In other embodiments, the cured material maycomprise a growth factor, such as for example a vascular endothelialgrowth factor (VEGF) or a fibroblastic growth factor.

The broadly described article of the disclosure may contain multiplechannels, e.g., in an arrangement in which at least some of the multiplechannels are in cell-signaling proximity to one another, and wherein thearticle comprises a cell attachment signal agent such asarginylglycylaspartic acid.

The molded body of the broadly described article of the disclosure maycomprise material that is permeable to cellular nutrients when cells aredisposed in the channel(s) of the three-dimensional cell growthcontainment article.

In various embodiments of the broadly described article of thedisclosure, the molded body comprises hydrogel material. The hydrogelmaterial may be ionically cross-linked. The hydrogel material maycomprise an alginate.

In various embodiments of the broadly described article of thedisclosure, at least one of the channel(s) of the article contains fillmaterial comprising at least one of monomer, prepolymer, cells, growthfactors, nano materials, small molecule drugs, antibodies, proteins,nucleic acids, nutrients, tracers, bacteria, viruses, and gas-producingsubstances. For example, the fill material may comprise a biologicalmaterial, which may be naturally occurring biological material and/orsynthetic biological material. The biological material may comprise atleast one of cells, antibodies, proteins, nucleic acids, bacteria, andviruses.

The broadly described article of the disclosure in various embodimentsmay include fill material comprising cells in the matrix material. Thecells may comprise suspension cells and/or the cells may compriseadherent cells affixed to interior surfaces of the channel(s) containingthe matrix material. The cells may comprise hormone secreting cells,e.g., wherein the cells are of one or more cell types of corticotropes,gonadotropes, thyrotropes, lactotropes, somatotropes, magnocellularneurosecretory, thyroid epithelial, parafollicular, parathyroid, adrenalgland, macula densa kidney, Leydig, granulosa lutein, theca lutein,peripolar kidney, and mesangial kidney cells. The cells may compriseislets of Langerhans, e.g. of one or more cell types of alpha cells,beta cells, delta cells, PP cells, and Epsilon cells. The cells maycomprise liver cells, e.g., one or more cell types of hepatocytes,stellate cells, Kupfer cells, and liver endothelial cells. The cells maycomprise peptide hormone secreting cells, e.g., of one or more celltypes of alpha cells, beta cells, corticotropic cells, delta cells,gonadotropin cells, gastric chief cells, lactotropic cells,parafollicular cells, parathyroid cells, somatotropic cells, andthyrotropic cells.

The broadly described article of the disclosure may in variousembodiments comprise a matrix material, as for example matrix materialcomprising cross-linked alginate, that further comprises a cellattachment signal agent, e.g., arginylglycylaspartic acid. In otherembodiments, the matrix material may comprise a growth factor, such asfor example a vascular endothelial growth factor (VEGF) or afibroblastic growth factor.

The molded body of the broadly described article of the disclosure mayin various embodiments comprise polyacrylamide. The molded body may forexample comprise polyacrylamide and cross-linked alginate.

The broadly described article of the disclosure in a form amenable todeployment or otherwise in use may be constituted as containing athree-dimensional cell population in the matrix material.

The broadly described article of the disclosure and the broadlydescribed method of the disclosure encompass, in various embodiments, aretrievable three-dimensional cell growth containment article in theform of an insulin production device that is introducible in the body ofa subject, e.g., by injection, or other suitable modality ofadministration. The article in such application may be constructed as ahollow, multi-layered implant that contains a population of islet cellsfor production of insulin, and is sufficiently permeable for insulin,glucose, and oxygen exchange in the body of the (human or veterinary)subject without allowing antibodies to reach islet cells.

Such insulin production device may include a hollow core in which isletcells suspended in alginate are provided, with RGD for effecting celladhesion. Such cell-containing core may be circumscribed by analginate-PA layer for stability, with an outermost layer of high-Galginate, and VEGF-C for vascularization. The outermost layer therebyprovides an immunoprotective layer in the structure, and the isletfunction is supported by the high-G alginate, the presence of RGD,VEGF-C on the surface for vascularization, and CaO₂ to generate oxygen.

It will therefore be appreciated that the size, form, and configurationof the channels, passages, and compartments of the three-dimensionalcell growth containment article may be selectively varied to provide adesired character and extent of three-dimensional cell growth in thearticle, as necessary or desirable in a given application of thearticle.

Referring now to the drawings, FIG. 1 is a top plan view of ahalf-section of a separable mold body usefully employed for forming athree-dimensional cell growth containment article in accordance with oneembodiment of the present disclosure.

As illustrated, the mold 10 includes a half-section mold body 12 shownin top plan view, of generally rectangular configuration, including acorrespondingly rectangular cavity 14 therein which in the illustratedplan view surrounds the cavity in the interior volume of thehalf-section mold body. The half-section mold body illustrated isoverlaid with a sacrificial core element 16, being reposed on the flatsurface of the half-section mold body, which is engageable with a secondhalf-section mold body (not shown) of corresponding shape to thehalf-section mold body illustrated, which may be disposed as a lowerhalf-section of the mold. The upper half-section of the mold issymmetrically formed with half-section mold body 12, being inverted withrespect to such half-section mold body so that facing flat surfaces ofthe two half-sections are made in face-to-face contact with one anotherto enclose the rectangular cavity 14, with the sacrificial core element16 being compressively retained between the respective half-sections ofthe mold body.

The respective mold body half-sections may be formed with inlet andoutlet ports (not shown in FIG. 1 ), for introduction of monomer orpre-polymer into the cavity 14 between the respective half-sections ofthe mold, following which the respective inlet and outlet ports may beclosed, and the monomer or pre-polymer composition in the cavity 14 maybe subjected to suitable curing conditions, e.g., elevated temperatureconditions, anaerobic conditions, or other conditions that may effectreaction and curing of the monomer or pre-polymer into a cured mass.

After the monomer or pre-polymer has been reacted and cured to acorresponding mass defined by the bounding walls of the cavity 14, theseparable half-sections of the mold body may be separated from oneanother to yield a unitary mass of product polymer, having thesacrificial core element 16 extending therethrough. The sacrificial coreelement 16 may then be removed by any appropriate removal conditions, toprovide a corresponding opening in the unitary mass of product polymer.For example, the sacrificial core element 16 may be a material that isamenable to removal from the cured mass of polymer, by grasping of oneextended in of the sacrificial core element and intentionally drawingsame out of the cured polymer mass to yield the through-bore opening 20in the resulting polymer article as the three-dimensional cell growthcontainment article 18.

As another alternative, the sacrificial core elements 16 may be formedof a material that is soluble in a suitable solvent medium which whencontacted with the cured mass of polymer leaches away the sacrificialcore element to leave the aforementioned through-bore passage in thecured mass of polymer, thereby yielding a three-dimensional cell growthcontainment article, having a central bore passage within which acellular population may be introduced for subsequent growth therein.

In the aforementioned method, the half-sections of the mold body may beformed of any suitable material such as for example metal, ceramic,composite material, or other suitable material that is compatible withthe monomer or prepolymer composition that is molded in the cavity ofthe mold body. The sacrificial core element 16 in such method may bealigned in the center of the cavity by applying tension on both ends asthe respective half-sections of the mold body are brought intoregistration alignment with one another. In such manner, the sacrificialcore element provides a structure, which when removed, generates ahollow core passage in the product molded body. The hollow core passagethen can be filled with cells, as mentioned above, e.g., in combinationwith a suitable scaffold material, such as another monomer orpre-polymer suitable for such purpose. The cellular population then maybe fixed in position by curing of the other monomer or pre-polymerintroduced with the cell population into the hollow core passage of themolded article. Alternatively, the monomer or prepolymer may beintroduced into the hollow core passage in advance of introducing thecell population thereto, so that a matrix is formed in the hollow corepassage, into which the cell population may be introduced.

FIG. 2 illustrates the removal of the sacrificial core element 16 fromthe molded body (polymer structure) to form the through-bore opening 20as a hollow core passage in the polymer structure of the molded body inthe formation of the three-dimensional cell growth containment article18. The hollow core passage then can be provided with a cellularpopulation introduced into the passage together with any suitableassociated material such as scaffolding, cellular nutrients, etc.

The mold that is utilized for forming the three-dimensional cell growthcontainment article can be designed in any of widely varying ways.

FIG. 3 depicts cross sections of polymer structures A-G that may befabricated with correspondingly configured molds. With the exception ofcross-section D, of the polymer structures are formed so as to provideinterior passages in which the cellular population may be disposed. Thecross-section D illustrates a hemi-cylindrical configuration that may becombined with other molded elements to provide one or more interiorpassages. For example, the hemi-cylindrical molded body of cross-sectionD may be engaged with a U-shaped channel member secured at itsextremities to the flat face of the molded body of cross-section D, tothereby form an enclosed interior volume through the resulting compositestructure. In addition, in molded bodies having multiple interiorpassages, the molded body may be formed of a permeable material allowinginfusion of cellular nutrients from one passage containing same toanother passage containing the cellular population, in order to maintainviability of the cellular population. In like manner, the molded bodymay contain an interior passage that is arranged to receive secretedmaterials, such as secreted therapeutic proteins, as well as wasteproducts such as cellular degradation products, from the cellularpopulation in another interior passage of the molded body.

Thus, the molded body may be provided with multiple compartments, inwhich each compartment can be at least partially filled with variouscomponents, including, without limitation, monomer, pre-polymer, cells,growth factors, nanomaterials, small molecule drugs, antibodies,proteins, nucleic acids, nutrients, tracers, bacteria, viruses,gas-producing substances, and compatible combinations of 2 or more ofthe foregoing.

In a specific implementation, the three-dimensional cell growthcontainment article is utilized to support a population of islet cellsin one or more passages in the article, for the prevention or treatmentof diabetes. For such purpose, the molded body may be employed to forman implant containing islet cells within a reservoir that isencapsulated by a core of polymer in the molded body, with such polymercomprising a biocompatible material capable of maintaining viability ofthe cells in the molded article.

The three-dimensional cell growth containment article in otherapplications may be utilized to encapsulate multiple cellular typeswithin a polymeric matrix with controlled spatial arrangement. Cellulartypes may for example include suspension cells and adherent cells.Specific examples of cells that may be deployed in three-dimensionalcell growth containment articles of the present disclosure include,without limitation, hormone secreting cells such as corticotropes,gonadotropes, thyrotropes, lactotropes, somatotropes, magnocellularneurosecretory cells, thyroid epithelial cells, parafollicular cells,parathyroid cells, adrenal gland cells, macula densa cells of thekidney, Leydig cells, granulosa lutein cells, theca lutein cells,peripolar cells of kidney, and mesangial cells of kidney. Langerhansislet cells may be provided in the passages of the three-dimensionalcell growth containment articles of the present disclosure, includingalpha cells, beta cells, delta cells, PP cells, epsilon cells, andcombinations thereof. Additional cell types include liver cells, such ashepatocytes, stellate cells, Kupffer cells, liver endothelial cells, andcombinations of two or more of the foregoing. Still further cell typesinclude peptide hormone secreting cells, including alpha cells, betacells, corticotropic cells, delta cells, gonadotropic cells, gastricchief cells, lactotropic cells, parafollicular cells, parathyroid cells,somatotropic cells, and thyrotropic cells, and combinations of two ormore of the foregoing.

Monomers that are used to prepare the polymeric matrix in thethree-dimensional cell growth containment articles of the presentdisclosure may be of any suitable type, and may for example includealginates, cellulose, chitosan, carboxymethylcellulose, acrylate,methacrylates, and acrylamides. Nonlimiting examples of acrylates andmethacrylates include polyethylene glycol (PEG) diacrylate and PEGdimethacrylate. In particularly advantageous embodiments, the PEG chainmay have a molecular weight in a range of from 1,000 to 5,000 (numberaverage molecular weight). In one preferred embodiment, the alginate hasa G:M ratio greater than 1.5. Other nonlimiting examples of polymerssuitable for formation of the matrix include poly lactic co-glycolicacid (PLGA), polycaprolactone, PEG, polylactide, polyglycolide,ethylene-vinyl acetate copolymer, and combinations of two or more of theforegoing.

Polymers utilized as support or scaffolding material in thethree-dimensional cell growth containment article may be of any suitabletype, and may for example be immunoprotective in character to supportcellular proliferation and survival. In various implementations, thepolymer may be chemically or physically modified to support the specificcells being incorporated in the cell growth containment article. Forexample, adherent cell lines may be utilized in combination withmonomers and or prepolymers that contain a cell attachment signal, suchas arginylglycylaspartic acid (RGD). In other implementations, cells maybe present together with a growth factor, such as for example a vascularendothelial growth factor (VEGF) or a fibroblastic growth factor. Instill other implementations, the cells may be grown in suspension in thethree-dimensional cell growth containment article.

In various embodiments, the molded article of the three-dimensional cellgrowth containment structure may be formed with multiple compartments,in which cells within a scaffolding precursor are injected into a firstcompartment and a biological agent within a scaffolding precursor isinjected into a second compartment. The biological agent may for exampleinclude any of growth factors, nanomaterials, small molecule drugs,antibodies, proteins, nucleic acids, nutrients, tracers, bacteria,viruses, gas-producing substances, and combinations of two or more ofthe foregoing. In such manner, the biological agent may be controllablydelivered to the cells or kept in close spatial proximity withoutcontacting the cells. As a specific example, paracrine signaling may beprovided by such type of arrangement. As another specific example,multiple compartments in the cell growth containment structure maycontain cells of different types that are permitted to grow close toeach other, e.g., to enable cell signaling and resultant behaviors to beevaluated.

The features and advantages of the present disclosure are more fullyappreciated with reference to the following non-limiting examples.

Example 1

Engineered Mold for Producing a Cylindrical Polymer with a Hollow Core

FIG. 4 is a digital camera image of an aluminum mold that was used toprepare a cylindrical cell growth containment article with a hollowcore. FIG. 5 shows CAD drawings of the mold design and FIG. 6 showsdigital camera images of the resulting mold.

A mold was fabricated for filling with selected monomer or prepolymer,as illustrated in FIG. 4 . By the use of such mold, a cylindricalpolymeric structure with hollow core can be produced, wherein the hollowcore provides an interior passage suitable for filling with any of avariety of cellular compositions. The mold was formed of aluminum, witheach of the separable half-sections of the mold including an engravedtrough arranged to accommodate a sacrificial core element. Thesacrificial core element comprised a cord of 1 mm diameter, and theengraved trough measured 2 mm in diameter. The cord was placed betweenthe two aluminum mold half-sections and the mold half-sections weremated with one another, with tensioning of the sacrificial cord prior totight securement of the half-sections of the mold to one another. Byapplying adequate tension, the sacrificial cord was centrally alignedwithin the hollow cylindrical trough formed by the respective engravedtroughs of the mated half-sections of the mold.

As shown in FIG. 4 , the mold featured an injection port in the upperhalf-section thereof, which is illustrated with a syringe injector beingcoupled to such injection port, for introduction of polymer or othercurable material into the mold cavity from the syringe injector. FIG. 5shows the CAD drawings of the mold, in which each mold half-section hasa longitudinal cavity formed therein so that when the half-sections ofthe mold are mated with one another, a corresponding cavity is formedfor injection of the polymer or other curable material, to form themolded body of the three-dimensional cell growth containment structure.FIG. 6 shows the open mold half-sections in side-by-side relation to oneanother, showing the central cavity (fill chamber) and a space at theends of the half-sections for the sacrificial core element, and theclosed mold with the injection port being identified by the arrow in thelower image of the figure.

Example 2

Production of a Structure Comprising a Polymeric Hollow Cylindrical Core

A mold of the type described in Example 1 was used to produce acylindrical structure with a hollow core, using a sacrificial threadelement. The aluminum mold was assembled and a monomeric composition wasinjected into the mold cavity. The monomeric composition comprised 2 mLof acrylamide solution (40% acrylamide in water at anacrylamide:bis-acrylamide ratio of 19:1), 4 mL of 3 wt. % alginate, 100μL of 10 wt. % ammonium persulfate (APS), and 10 μL ofN,N,N′,N′-tetramethylethylenediamine (TEMED). After injection of themonomeric composition, the mold was permitted to remain at roomtemperature for at least 15 minutes, during which time the acrylamidepolymerized. After at least 15 minutes, the two portions of the moldwere opened and the polymerized cylinder containing the sacrificial corewas removed from the mold, and the sacrificial core element was removed,resulting in a hollow polymeric cylinder. FIG. 7 is a microscopic imageof the hollow polymeric cylinder comprising polyacrylamide andnon-cross-linked alginate with a hollow core.

Next, the hollow cylinder was placed in a cross-linking solution of 50mM calcium chloride or 50 mM aluminum chloride for 40 minutes tocross-link the alginate in the cylinder.

FIG. 8 is a microscopic image of a cross-section of the cylindricalpolymer structure comprising polyacrylamide and alginate, and having ahollow core passage with a diameter of 1 mm.

FIG. 9 is a digital camera image of the cylindrical polymer structure ofthe polyacrylamide/alginate polymer containing a hollow core passage, inwhich the structure was 12 mm in length with an outer diameter of 2 mm.

FIG. 10 summarizes the steps involved in forming the hollow corecylindrical polymer structure of the three-dimensional cell growthcontainment article. Step 1 involves injection of theacrylamide/alginate solution into the mold and polymerization thereof.Step 2 involves removal of the hollow polyacrylamide (PA)/alginatemolded body, in which PA is polymerized, and the alginate remainsunpolymerized. Step 3 involves placement of the hollow core cylindricalpolymer structure into a cross-linking solution of either AlCl₃ orCaCl₂) and allowing the alginate to cure.

Example 3

Production of a Hollow Core Molded Body Article with a Three-DimensionalCellular Scaffold in the Hollow Core Passage

A hollow core cylindrical polymer molded body was formed as described inExample 2, and such molded body, having a 2 mm outer diameter, 1 mmhollow core passage diameter, and length of 12 mm, was placed in 100%ethanol to sterilize the structure. Next, the sterilized molded body wasplaced in a cellular medium comprising RPMI-1640 supplemented with 10vol % fetal bovine serum (FBS) for at least 2 hours. During itsimmersion in ethanol, the molded body shrinks due to expulsion of watertherefrom, but it readily rehydrates when placed in the cellular medium.A solution was prepared comprising 9 g of 2% alginate in 150 mM NaCl (1g of 150 mM NaCl).

Next, a B lymphocyte cell line of Raji cells was suspended in RPMI-1640cell medium and subsequently mixed with the alginate solution at a ratioof 3:1 of alginate:cells. The final concentration of cells in thismixture was nearly 5×10⁵ cells/mL. The hollow molded article then wasinjected with approximately 100 μL of such mixture, and the articlecontaining the mixture was incubated in 50 mM CaCl₂ in 150 mM NaCl forapproximately 15 minutes to effect cross-linking of the core. Theresulting cell growth containment article then was transferred to cellculture medium and placed at 37° C. and 5% CO₂ to permit cell growth.

FIG. 11 shows the sequence of the foregoing steps for producing athree-dimensional cell growth containment article including a core ofcells in a three-dimensional scaffold. Step 1 involves placement of thehollow molded device in ethanol for 20 minutes to effect sterilization,followed by conditioning in cell medium. Step 2 involves filling themolded hollow body with a mixture of cells and alginate using a needle,and Step 3 involves cross-linking the alginate used as cellularscaffolding in the molded article hollow passage, by placing the moldedarticle into cross-linking solution (either aluminum chloride or calciumchloride solution), followed by culturing at 37° C. and 5% CO₂ ambient.

FIG. 12 shows the results after culturing the Raji cells in thethree-dimensional cell growth containment article for 3 days, influorescence images of Raji cells in the interior of the article, asstained with a Live-Dead staining kit. The images show fluorescence fromcalcein AM (green fluorescence), which indicated that the cells werealive. Conversely, no fluorescence resulted from ethidium (whichproduces red fluorescence), which indicated minimal dead cells in thecell growth containment article.

FIG. 13 shows beta cell line RIN-5F cells alive and proliferating in thethree-dimensional cell growth containment article after 25 days. Thecell growth containment article contained alginate cross-linked withcalcium chloride, as the support matrix for the cells. Fluorescenceimages are shown of the RIN-5F cells stained with Live-Dead stainingkit, in the interior of the cell growth containment article. The imagesshow fluorescence from calcein AM (green), which indicates the cells arealive. Conversely, no fluorescence was observed resulting from ethidium(red), thereby indicating minimal dead cells.

FIG. 14 shows the steps for loading a cell mixture into thethree-dimensional cell growth containment article, using a solutionrepresenting a surrogate for cells for purposes of illustration. Step 1illustrates a pipette tip being loaded with 1:3 trypan blue and 2.22%alginate (pre-warmed). In Step 2, the pipette tip is inserted into thethree-dimensional cell growth containment article passage. In Step 3,the three-dimensional cell growth containment article is carefullyloaded with the alginate-trypan blue solution. In Step 4, thethree-dimensional cell growth containment article has been loaded withthe hollow core passage being filled. In Step 5, the three-dimensionalcell growth containment article in loaded condition is ready fordeployment.

While the disclosure has been set forth herein in reference to specificaspects, features and illustrative embodiments, it will be appreciatedthat the utility of the disclosure is not thus limited, but ratherextends to and encompasses numerous other variations, modifications andalternative embodiments, as will suggest themselves to those of ordinaryskill in the field of the present disclosure, based on the descriptionherein. Correspondingly, the disclosure as hereinafter claimed isintended to be broadly construed and interpreted, as including all suchvariations, modifications and alternative embodiments, within its spiritand scope.

What is claimed is:
 1. A method of making a three-dimensional cellgrowth containment article, said method comprising: engaging mold bodyportions with one another to constitute a separable mold body defining amold cavity therewithin between the engaged mold body portions with atleast one sacrificial channelizing element compressively retainedbetween the engaged mold body portions so that the sacrificialchannelizing element spans the mold cavity; introducing a curable mediuminto the mold cavity so that the curable medium fills the mold cavity toa predetermined extent, and contacts and circumscribes the sacrificialchannelizing element(s) in a bulk volume of the curable medium, thecurable medium comprising a polymerizable monomer or prepolymer, and across-linkable material; polymerizing the polymerizable monomer orprepolymer of the curable medium to form a polymerized articlecomprising the cross-linkable material and containing the at least onesacrificial channelizing element; removing the polymerized article fromthe mold cavity and removing the sacrificial channelizing element(s)from the polymerized article so that the polymerized article ischannelized by the removed sacrificial channelizing element(s);cross-linking the cross-linkable material in the channelized polymerizedarticle; introducing a fill material to one or more channel(s) in thechannelized polymerized article after said cross-linking, the fillmaterial comprising cells and a curable cell support matrix material;curing the curable cell support matrix material in the channel(s) toform a cured matrix supporting the cells in the channel(s); andcontacting the cells supported in the cured matrix in the channel(s)with a cell culture medium, to yield the three-dimensional cell growthcontainment article containing channel(s) for three-dimensional cellgrowth therein.
 2. The method of claim 1, wherein the curable mediumcomprises: an alginate; cellulose; chitosan; collagen; fibrin;glycosaminoglycan; carboxymethylcellulose; a monomer or pre-polymercontaining a cell attachment signal agent; acrylate; methacrylate;acrylamide; material that is permeable to cellular nutrients; growthfactor; hydrogel material; or a combination of two or more of theforegoing.
 3. The method of claim 1, wherein the curable medium whencured comprises: polylactic-co-glycolic acid (PLGA); polycaprolactone;polyethylene glycol; polylactide; polyglycolide; ethylene-vinyl acetatecopolymer; an immunoprotective polymer, or a combination of two or moreof the foregoing.
 4. The method of claim 1, wherein thethree-dimensional cell growth containment article contains multiplechannels, and wherein the curable medium comprises arginylglycylasparticacid.
 5. The method of claim 1, wherein the curable medium when curedcomprises an alginate cross-linked with calcium (Ca⁺²) cations.
 6. Themethod of claim 1, wherein the mold body portions are formed ofdegradable sacrificial material, and the method further comprisesdegrading the degradable sacrificial material after polymerizing thepolymerizable monomer or prepolymer of the curable medium, wherein thedegrading comprises at least one of chemically decomposing thedegradable sacrificial material, volatilizing the degradable sacrificialmaterial, dissolving the degradable sacrificial material, irradiatingthe degradable sacrificial material with degradative radiation, andthermally decomposing the degradable sacrificial material.
 7. The methodof claim 1, wherein the sacrificial channelizing element comprises afiber, filament, or cord that is maintained in tension across itsspanning extent.
 8. The method of claim 1, wherein said fill materialcomprises at least one of monomer, prepolymer, cells, growth factors,nano materials, drugs, antibodies, proteins, nucleic acids, nutrients,tracers, bacteria, viruses, and gas-producing substances.
 9. The methodof claim 1, wherein said cells are of one or more cell types ofcorticotropes, gonadotropes, thyrotropes, lactotropes, somatotropes,magnocellular neurosecretory, thyroid epithelial, parafollicular,parathyroid, adrenal gland, macula densa kidney, Leydig, granulosalutein, theca lutein, peripolar kidney, and mesangial kidney cells. 10.The method of claim 1, wherein the curable material comprisesacrylamide, water, alginate, ammonium persulfate, andtetramethylenediamine (TEMED).
 11. The method of claim 1, wherein saidfill material comprises adherent cells in combination with monomers orpre-polymers that contain a cell attachment signal agent.
 12. The methodof claim 1, wherein the curable cell support matrix material comprisesalginate, and curing thereof comprises contacting with a cross-linkingsolution of calcium dichloride or aluminum trichloride to cross-link thealginate, so that the cells are supported in a cured matrix ofcross-linked alginate.